Nef is a major determinant of pathogenesis; both human and simian immunodeficiency viruses (HIV/SIV) show an attenuated phenotype when the accessory protein Nef is deleted. In vitro, both HIV and SIV Nef demonstrate several activities which include enhancement of infectivity, CD4 and MHCI downregulation and the activation of p21-activated protein kinase 2 (Pak-2) among others. Interestingly, Nef achieves all these activities in the face of high sequence divergence. It has been suggested that the complex biology of Nef is regulated through conformational changes induced by its cellular location and specific protein-protein interactions during cell traffic. In order to understand the importance of each of the Nef-mediated activities, in particular the activation of the host cell kinase Pak-2 to HIV/SIV pathogenesis, we have identified a surface on the subtype B Nef molecule that is specific for this activity. However, analysis of the amino acid sequence of subtype E Nef sequences indicated that dramatically different amino acid substitutions in this surface that completely abolish the ability of subtype B Nef to activate Pak-2 result in fully functional subtype E Nefs. Our biochemical analysis together with in silico modeling of Nef structure resulted in the identification of compensatory mutations that restore Nef activity. As part of this grant we first propose to identify these compensatory mutations in Nef from additional HIV-1 subtypes to fully define this surface and secondly to extend these observations to SIV Nef. We propose to identify alternative structures of this surface as it relates to variation between subtypes and even between strains. We will compile sequences in all available databases and translate our observations by generating mutated versions of the prototypical SF2 Nef that reflect naturally occurring sequences. The effect of these substitutions to the Nef-mediated activities, particularly the activation of Pak-2, will be correlated with changes in the tridimensional structure of Nef. The studies proposed here will help elucidate the structural requirements for the activation of Pak-2 by Nef and will serve as the basis for future in vivo experiments aimed at characterizing its functional significance. [unreadable] [unreadable] [unreadable]